ACCELERATED COMMUNICATION Deorphanization of GPRC6A: A Promiscuous L- -Amino Acid Receptor with Preference for Basic Amino Acids

One of the most important tasks of molecular pharmacology is the deorphanization of the large number of G-protein-coupled receptors with unidentified endogenous agonists. We recently reported the cloning and analysis of expression of a novel human family C G-protein-coupled receptor, termed hGPRC6A. To identify agonists at this orphan receptor, we faced the challenges of achieving surface expression in mammalian cell lines and establishing an appropriate functional assay. Generating a chimeric receptor construct, h6A/5.24, containing the ligand binding amino-terminal domain (ATD) of hGPRC6A with the signal transducing transmembrane and C terminus of the homologous goldfish 5.24 receptor allowed us to overcome these obstacles. Homology modeling of the hGPRC6A ATD based on the crystal structure of the metabotropic glutamate receptor subtype 1 predicted interaction with -amino acids and was employed to rationally select potential ligands. Measurement of Ca -dependent chloride currents in Xenopus laevis oocytes facilitated the deorphanization of h6A/ 5.24 and identification of L-amino acids as agonists. The most active agonists were basic L-amino acids, L-Arg, L-Lys, and L-ornithine, suggesting that these may function as endogenous signaling molecules. Measurement of intracellular calcium in tsA cells expressing h6A/5.24 allowed determination of EC50 values, which confirmed the agonist preferences observed in oocytes. Cloning, cell surface expression and deorphanization of the mouse ortholog further reinforces the assignment of the agonist preferences of hGPRC6A. This study demonstrates the utility of a chimeric receptor approach in combination with molecular modeling, for elucidating agonist interaction with GPRC6A, a novel family C G-protein-coupled receptor. With the complete human genome sequence accessible, the identification of novel genes has been greatly facilitated, which has led to the prediction of a large number of orphan receptor genes (Venter et al., 2001; Wise et al., 2004). This has sparked intense interest, particularly within the field of G-protein-coupled receptors (GPCRs), which represent about 50% of current drug targets. Deorphanizing receptors (i.e., identifying one or more endogenous agonists) has been difficult, and the physiological relevance of many of these potential drug targets remains unknown (Wise et al., 2004). We identified the human GPCR, family C, subtype 6A (hGPRC6A) receptor by homology searches in sequence databases and subsequently cloned a putative human orphan GPCR (Wellendorph and Bräuner-Osborne, 2004). Cloning revealed the existence of three different splice variant forms, verified by the exon-intron organization of the gene for hGPRC6A, mapping to chromosome 6q22.31 (GenBank accession numbers AY435125, AY435126, and AY435127). Reverse transcription-PCR analysis showed that the longest isoform of GPRC6A was expressed at the highest level in human tissues, and an ortholog of that form was also found in mice (GenBank accession number AY101365). GPRC6A displays 45% amino acid sequence identity with the goldfish This work was supported by the Danish Medical Research Council, Apotekerfonden of 1991, the Lundbeck Foundation, and EU grant HPAW-CT2002-80057 (to P.W.). □S The online version of this article (available at http://molpharm. aspetjournals.org) contains supplemental material. Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org. doi:10.1124/mol.104.007559. ABBREVIATIONS: GPCR, G-protein-coupled receptor; GPRC6A, G-protein-coupled receptor, family C, group 6, subtype A; h, human; m, murine; mGlu, metabotropic glutamate; ATD, amino-terminal domain; PCR, polymerase chain reaction; BAPTA, 1,2-bis(2-aminophenoxy)-ethaneN,N,N ,N -tetraacetic acid; AM, acetoxymethyl ester; Orn, ornithine; Cit, citrulline; 7TM, seven transmembrane domain. 0026-895X/05/6703-589–597$20.00 MOLECULAR PHARMACOLOGY Vol. 67, No. 3 Copyright © 2005 The American Society for Pharmacology and Experimental Therapeutics 7559/1194973 Mol Pharmacol 67:589–597, 2005 Printed in U.S.A. 589 http://molpharm.aspetjournals.org/content/suppl/2004/12/02/mol.104.007559.DC1 Supplemental material to this article can be found at: at A PE T Jornals on A uust 8, 2017 m oharm .aspeurnals.org D ow nladed from at A PE T Jornals on A uust 8, 2017 m oharm .aspeurnals.org D ow nladed from at A PE T Jornals on A uust 8, 2017 m oharm .aspeurnals.org D ow nladed from at A PE T Jornals on A uust 8, 2017 m oharm .aspeurnals.org D ow nladed from at A PE T Jornals on A uust 8, 2017 m oharm .aspeurnals.org D ow nladed from at A PE T Jornals on A uust 8, 2017 m oharm .aspeurnals.org D ow nladed from at A PE T Jornals on A uust 8, 2017 m oharm .aspeurnals.org D ow nladed from at A PE T Jornals on A uust 8, 2017 m oharm .aspeurnals.org D ow nladed from at A PE T Jornals on A uust 8, 2017 m oharm .aspeurnals.org D ow nladed from at A PE T Jornals on A uust 8, 2017 m oharm .aspeurnals.org D ow nladed from odorant receptor 5.24 (Speca et al., 1999), 34% identity with the human calcium-sensing receptor (Brown, 1999), and 28% identity with the human taste receptor T1R1 (Nelson et al., 2001; Wellendorph and Bräuner-Osborne, 2004). This places hGPRC6A in family C of GPCRs, which also includes the metabotropic glutamate (mGlu1–8) receptors (Pin and Duvoisin, 1995), GABAB1–2 (Möhler and Fritschy, 1999), and some orphan and pheromone receptors (Cheng and Lotan, 1998; Bräuner-Osborne and Krogsgaard-Larsen, 2000; Robbins et al., 2000; Bräuner-Osborne et al., 2001; Calver et al., 2003). Family C GPCRs are distinguished from other GPCR superfamilies by an unusually large amino-terminal domain (ATD), consisting of a globular ligand binding bi-lobular structure (lobe I and II) connected by a hinge region (Kunishima et al., 2000). Based on the crystal structure of glutamate bound to the ATD of mGlu1 (PDB code 1EWK), we have generated a three-dimensional homology model of GPRC6A, suggesting that the endogenous agonist for GPRC6A is an -amino acid. The lack of cell surface expression of the hGPRC6A protein in mammalian cell lines (Wellendorph and Bräuner-Osborne, 2004) hampered the development of a functional pharmacological assay to examine this prediction. To overcome this obstacle, we have constructed chimeric receptors of hGPRC6A and the close functional 5.24. The chimera composed of the ATD of hGPRC6A, and the 7TM domain and carboxyl terminus of 5.24 (h6A/5.24), confirmed that L-amino acids can bind to the hGPRC6A ATD and activate the 5.24 dependent signal transduction pathways when expressed in Xenopus laevis oocytes or tsA cells, thus supporting our proposed model. We further document these findings by cloning and deorphanization of the native murine homolog of GPRC6A (mGPRC6A) and providing the first steps toward unraveling the function of GPRC6A. Materials and Methods Homology Modeling and Ligand Docking. A homology model of the ligand-binding domain of GPRC6A was built from the protein subsequence (1EWK chain A) corresponding to the crystallized extracellular domain of the mGlu1 receptor (Kunishima et al., 2000), with the aid of the Polish Bioinformatics metaserver (Ginalski et al., 2003) and Easypred (Lambert et al., 2002) including MODELER (Marti-Renom et al., 2000). Based on a high degree of conservation of the region binding the -amino acid moiety of L-Glu in the mGlu1 receptor and the location of acidic residues, tri-ionized L-Lys was modeled into the binding site by a combination of manual docking and restrained minimization, followed by Monte Carlo conformational searching of ligand and side chains with OPLS-AA in Macromodel 8.1 and Prime 1.1 (Schrödinger Inc., Portland, OR) and analysis of receptor water placement using Glide 2.5 (Schrödinger Inc.). For details, see Supplemental data. Cloning of GPRC6A. Human GPRC6A was cloned in our laboratory as recently reported (GenBank accession number AY435125) (Wellendorph and Bräuner-Osborne, 2004). Full-length mouse GPRC6A was cloned by nested PCR according to the following protocol (kindly provided by T. M. Strom, Technical University Munich, Germany). The first round of PCR was carried out on cDNA generated from 17-day-old embryos (BD Biosciences, Palo Alto, CA) using Pfu polymerase (Stratagene, La Jolla, CA), forward primer 5 -gctcttaataaccctcatgaac-3 and reverse primer 5 -aaagtaaatacacaatttgcagc-3 (20 cycles at 50°C annealing). The second PCR was performed with forward primer 5 -catgaactgagcaaatgagac-3 and reverse primer 5 -gaaacatctcactggggatc-3 (30 cycles at 50°C annealing). A single band of 2900 base pairs was purified (QIAquick Gel Extraction kit; QIAGEN, Hilden, Germany) and TOPO TA cloned into the pCR2.1 vector (Invitrogen, San Diego, CA) according to the manufacturer’s instructions. The obtained cDNA was fully sequenced (MWG Biotech, Ebersberg, Germany) and found to be identical at the protein level with GenBank entry number AY101365. Construction of hGPRC6A and Goldfish 5.24 Chimeras. Chimeras were constructed containing the entire ATD of 5.24 and the 7TM and intracellular domains of hGPRC6A (5.24/h6A) and vice versa (h6A/5.24) by means of overlap extension PCR (Horton et al., 1989). The fusion site in 5.24/h6A was placed between amino acids 16 and 17 upstream of the predicted first transmembrane segment of hGPRC6A (Wellendorph and Bräuner-Osborne, 2004), in accordance with family C chimeras previously generated in our laboratory (Bräuner-Osborne et al., 1999b). The fusion site in h6A/5.24 was similarly constructed upstream of the first TM segment of 5.24 based on an alignment of hGPRC6A and 5.24 (Wellendorph and BräunerOsborne, 2004). All PCRs were performed with Pfu polymerase (Stratagene) following the manufacturer’s protocol. Chimeric receptor constructs were confirmed by DNA sequencing (MWG Biotech). Further details on construction of chimeras are available as Supplemental data. Site-Directed Mutagenesis of hGPRC6A and Chimera h6A/ 5.24. The 919–921RKR/AAA mutation in hGPRC6A and point mutations (S149A and T172A) in both hGPRC6A and chimera h6A/5.24 were introduced using the QuikChange mutagenesis kit (Stratagene). Mutations were confirmed by DNA sequencing (MWG Biotech). Epitope Tagging. For cellular expression studies, all receptor constructs were subcloned into a modified pEGFP-N1 vector (BD Biosciences) essentially as described previously (Pagano et al., 2001). In brief, the N-terminal signal peptides were replaced by the mGlu5 receptor signal peptide, because the latter is known to promote good receptor expression and proper release of the signal peptide. To allow detection of receptor surface expression by immunofluorescence, a c-myc epitope followed by an engineered MluI site was inserted immediately after the signal peptide of mGlu5, using a previously generated construct of hGPRC6A in this modified pEGFP-N1 vector (Wellendorph and Bräuner-Osborne, 2004), the cDNA was cut out by the flanking restriction enzymes MluI/NotI and replaced with either the goldfish receptor 5.24 cDNA or chimeric receptor cDNAs. For similar subcloning of mGPRC6A into pEGFP-N1, a MluI/NotI embraced receptor cDNA fragment lacking the native signal peptide was generated by PCR using forward primer 5 -cactcgacgcgttgtcataccccagatgac-3 and reverse primer 5 -cttcttctgcggccgcctcctaggaactcaatc-3 . The signal peptide was predicted by the program SignalP with a cleavage site between amino acid positions 20 and 21 (Nielsen et al., 1997). Cell Culture Work and Immunochemistry. Cell culturing of tsA (a transformed HEK 293 cell line) cells and quantification of receptor expression by means of an Amplex Red horseradish peroxidase-amplified enzyme-linked immunoassay (Molecular Probes, Leiden, The Netherlands) was carried out exactly as described previously (Wellendorph and Bräuner-Osborne, 2004). Quantification was accomplished by measuring fluorescence intensity (excitation at 530 nm/emission at 590 nm) on a NOVOstar microplate reader (BMG Labtechnologies, Offenburg, Germany). All data points were obtained in triplicate and confirmed in three independent experiments. Statistical significance was assessed using student’s t test. Oocyte Preparation and Injection. For expression in X. laevis oocytes, cDNAs were subcloned into the pGEMHE-3Z vector containing an upstream T7 promoter. Linearized plasmids were used to produce cRNAs with mMessage mMachine kits (Ambion, Austin, TX). Oocytes were surgically removed from mature female X. laevis frogs anesthetized in a 0.4% MS-222 (3-aminobenzoic acid ethyl ester) solution (Sigma-Aldrich, St. Louis, MO) for 10 to 15 min. To remove the follicle layer, the oocytes were subsequently placed in OR2 buffer (82.5 mM NaCl, 2.0 mM KCl, 1.0 mM MgCl2, and 5.0 mM HEPES, pH 7.5) including 0.5 mg/ml collagenase (type IA) (Sigma590 Wellendorph et al. at A PE T Jornals on A uust 8, 2017 m oharm .aspeurnals.org D ow nladed from Aldrich) for 2 to 3 h at room temperature. Healthy-looking stage V–VI oocytes were selected and the following day injected with cRNA (25–75 ng in 50 nl of water) and maintained in Modified Barth’s solution (88 mM NaCl, 1.0 mM KCl, 2.4 mM NaHCO3, 0.41 mM CaCl2, 0.82 mM MgSO4, 0.3 mM Ca(NO3)2, 15 mM HEPES, pH 7.5, 2% sodium pyruvate, 100 IU/ml penicillin, and 100 g/ml streptomycin) at 18°C. For calcium buffering experiments, oocytes were incubated for 30 min in OR2 buffer containing 100 M BAPTA-AM (Sigma-Aldrich). Electrophysiology. Whole-cell currents were recorded on oocytes 2 to 4 days after injection using two-electrode voltage clamp at 80 mV in normal frog Ringer’s solution (115 mM NaCl, 2.5 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, and 10 mM HEPES, pH 7.6). Recording pipettes were filled with 3 M KCl. Recordings were performed at ambient temperatures using OC-725C Oocyte Clamp amplifier (Warner Instruments, Hamden, CT) with a Digidata 1322A interface (Axon Instruments, Union City, CA). The pClamp7 suite of programs (Axon Instruments) was used for data acquisition. All tested compounds were purchased from Sigma-Aldrich. Measurement of Intracellular Calcium Levels. tsA cells were maintained and transfected as described previously (Wellendorph and Bräuner-Osborne, 2004) except that two million cells were plated in a 10-cm dish and transfected the following day with 8 g of plasmid DNA. The day after transfection, cells were split into polyD-lysine-coated black 96-well plates with clear bottoms (BD Biosciences). Two days after transfection, pharmacological activity was assessed by measurement of intracellular calcium levels essentially as described previously (Kuang et al., 2003). In brief, cells were washed with assay buffer (5.3 mM KCl, 0.44 mM KH2PO4, 4.2 mM NaHCO3, 138 mM NaCl, 0.34 mM Na2HPO4, 5.6 mM D-Glucose, 20 mM HEPES, 1 mM CaCl2, 1 mM MgCl2 and 1 mg/ml bovine serum albumin, pH 7.4) and preincubated for 2 h at 37°C in 100 l of assay buffer. The assay buffer was exchanged and cells were preincubated for another 2 h at 37°C. The assay buffer was then replaced with 50 l of assay buffer containing 6 M Fluo-4AM (Molecular Probes) and incubated for 1 h at room temperature in the dark. Finally, cells were washed three times with assay buffer without bovine serum albumin and then incubated with 150 l of assay buffer without bovine serum albumin for 30 min at room temperature in the dark. The cell plate was then transferred to a NOVOstar microplate reader, and responses were recorded at room temperature using excitation/emission wavelengths of 485 and 520 nm, respectively. Responses ( fluorescence units) were calculated as peak fluorescence after agonist addition subtracted fluorescence before agonist addition. Concentration-response curves were analyzed by nonlinear regression using Prism 4.0 (GraphPad Software, San Diego, CA). Pharmacological experiments were performed in triplicate and repeated in three independent experiments.